A COMPARISON OF THE RESPONSES TO ENVIRONMENTAL STRESS OF THE GRAM-POSITIVE BACTERIUM STAPHYLOCOCCUS XYLOSUS AND THE GRAM-NEGATIVE BACTERIUM HALOMONAS HALO BY ABDULRAHMAN ABDULLAH AL-HUMIANY (B. SC., MSC. THE UNIVERSITY OF KING ABDULAZIZ, JEDDAH, SAUDI ARABIA) THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN THE DEPARTMENT OF MOLECULAR BIOLOGY AND BIOTECHNOLOGY UNIVERSITY OF SHEFFIELD OCTOBER 1999 CONTENTS SUMMARY viii .................................................................................... DEDICATION ix .................................................................................. ACKNOWLEDGEMENTS x .................................................................. ABBREVIATIONS xi ............................................................................ CHAPTER 1: INTRODUCTION 1.1 THE HYPERSALINE ENVIRONMENT 1 .................................................... 1.1.1 THE GREAT SALT LAKE 2 ..................................................................... 1.1.2 THE DEAD SEA 2 .................................................................................... 1.1.3 HYPERSALINE LAKES IN ANTARCTICA 3 ............................................ 1.1.4 SABKHAS 3 ............................................................................................. 1.1.5 ALKALINE LAKES 4 ................................................................................ 1.1.6 MARINE SALTERNS 4 ............................................................................ 1.1.7 HYPERSALINE SOILS 5 ......................................................................... WHICH INHABIT SALINE ENVIRONMENTS 5 1.2 ORGANISMS ................... TO 10% (WN) SALINITIES 6 1.2.1 SEA WATER ............................................ (WN) SALINITIES 8 1.2.2 10-30% .................................................................. 30% (WN) SALINITIES 10 1.2.3 ABOVE ......................................................... 1.3 THE AVAILABILITY OF NUTRIENTS IN SALINE ENVIRONMENTS.... 11 1.4 THE TERMINOLOGY OF SALT ADAPTATION 13 ..................................... ACTIVITY (Aw) 13 1.4.1 WATER ...................................................................... 1.4.2 WATER POTENTIAL 14 .......................................................................... OSMOTIC PRESSURE 14 1.4.3 ....................................................................... 1.4.5 TURGOR PRESSURE 15 ........................................................................ 1.4.6 OSMOREGULATION 15 .......................................................................... 1.5 OSMOTIC STRESS AND THE ROLE OF COMPATIBLE SOLUTES.... 15 1.5.1 THE DIVERSITY OF COMPATIBLE SOLUTES 17 ................................. 1.5.2 THE MODE OF ACTION OF COMPATIBLE SOLUTES 26 ..................... 1.6 THE DIVERSITY OF HALOPHILIC AND HALOTOLERANT MICROORGANISMS 27 ................................................................................... 1.6.1 HETEROTROPHIC EUBACTERIA 28 ..................................................... 1.6.2 ARCHAEA 33 .......................................................................................... 1.6.3 CYANOBACTERIA 34 ............................................................................. 1.6.4 OTHER PHOTOTROPHIC EUBACTERIA 34 .......................................... 1.6.5 ALGAE 35 ................................................................................................ 1.7 AIMS OF PROJECT 36 ............................................................................... CHAPTER 2: MATERIALS AND METHODS 2.1 ISOLATION AND SELECTION OF ORGANISMS 37 ................................. AND GROWTH CONDITIONS 40 2.2 MEDIA .................................................. 2.2.1 MEDIA 40 ................................................................................................. CULTURES 42 2.2.2 BATCH ............................................................................ CULTURE 42 2.2.3 CONTINUOUS .................................................................. 2.2.4 PURIFICATIONAND MAINTENANCE OF THE ORGANISMS 43 .......... ii 2.2.5 EFFECT OF SODIUM CONCENTRATION ON GROWTH 44 ................. 2.2.6 EFFECT OF POTASSIUM CONCENTRATION ON GROWTH 44 .......... 2.2.8 EFFECT OF ON GROWTH 45 pH ........................................................... 2.2.9 EFFECT OF INHIBITORS ON GROWTH 45 ........................................... 2.3 METHODS USED TO IDENTIFY JDP14 45 ............................................... 2.3.1 MORPHOLOGY 46 .................................................................................. 2.3.2 MOTILITY ...........................................................................................47 2.3.3 CATALASE TEST 47 ............................................................................... 2.3.4 OXIDASE TEST 48 .................................................................................. 2.3.5 API STAPH KIT 48 ................................................................................... 2.4 MEASUREMENT OF RESPIRATION RATE 49 ......................................... 2.5 PROTEIN DETERMINATION 52 ................................................................ 2.6 PROLINE DETERMINATION 53 ................................................................. 2.7 GROWTH OF HALOMONAS HALO AND JDP14 ON CDM 53 .................. 2.8 EFFECT OF COMPATIBLE SOLUTES ON GROWTH AT 3M NaCL... 54 2.9 14C-GLYCINE BETAINE UPTAKE EXPERIMENTS 54 ............................... 2.9.1 MANUFACTURE OF 14C-GLYCINE BETAINE 54 ................................... 2.9.2 MEASUREMENT OF BETAINE UPTAKE 55 ........................................... 2.9.3 EFFECT OF INHIBITORS ON 14C-BETAINETRANSPORT 56 ............... 2.10 SILICONE OIL TECHNIQUE 56 ............................................................... 2.10.1 INTRACELLULARVOLUME (ICV) DETERMINATION 56 ..................... iii 2.10.2 MEASUREMENTOF MEMBRANE POTENTIAL (0T) 60 ..................... 2.10.3 EFFECT OF EXTERNAL PH 61 ............................................................ 2.10.4 EFFECT OF INHIBITORS 61 ................................................................. 2.11 EFFECT OF TEMPERATURE AND SALT ON ENZYME ACTIVITIES OF HALOMONAS HALO AND JDP14 63 ......................................................... 2.11.1 PREPARATION OF CELL FREE EXTRACT 63 .................................... 2.11.2 GENERAL ASSAY CONDITIONS 64 ..................................................... 2.11.3 FUMARASE (EC 4.2.1.2) 64 .................................................................. 2.11.4 MALATE DEHYDROGENASE(EC 1.1.1.37) 65 .................................... 2.11.5 ACID PHOSPHATASE 66 ...................................................................... 2.11.6 ß-GALACTOSIDASE(EC 3.2.1.23) 67 .................................................. CHAPTER 3: SALT TOLERANCE OF HALOMONAS HALO AND STAPHYLOCOCCUS XYLOSUS 3.1 INTRODUCTION 70 .................................................................................... 3.2 RESULTS 73 .............................................................................................. 3.2.1 ISOLATIONAND SELECTION OF ORGANISMS 73 ............................. 3.2.2 IDENTIFICATION OF JDP14 75 .............................................................. 3.2.3 GROWTH OF HALOMONAS HALO AND S. XYLOSUS AT DIFFERENT SALINITIES .........................................................................75 3.2.4 ELECTRON MICROGRAPHS OF S. XYLOSUS CELLS GROWN AT SALINITIES DIFFERENT .............................................................................76 3.2.5 EFFECT OF KCL ON GROWTH OF HALOMONAS HALO AND S. XYLOSUS 97 .................................................................................................... Iv 3.2.6 EFFECT OF KCL AND NaCl ON GROWTH OF HALOMONAS HALO AND S. XYLOSUS 97 ....................................................................................... 3.2.7 Na' REQUIREMENT FOR RESPIRATION IN HALOMONAS HALO AND S. XYLOSUS 102 ..................................................................................... 3.2.8 EFFECT OF KCL ON THE RESPIRATION RATE OF HALOMONAS HALO AND S. XYLOSUS 102 .......................................................................... 3.2.9 EFFECT OF NACL AND KCL ON THE RESPIRATION RATES OF HALOMONAS HALO AND S. XYLOSUS 103 ................................................... 3.3 DISCUSSION 109 ...................................................................................... CHAPTER 4: ROLE OF COMPATIBLE SOLUTES IN SALT TOLERANCE OF HALOMONAS AND STAPHYLOCOCCUS XYLOSUS 4.1 INTRODUCTION 115 .................................................................................. 4.2 RESULTS 118 ............................................................................................ 4.2.1 GROWTH OF S. XYLOSUS AND HALOMONAS HALO ON CHEMICALLY DEFINED MEDIUM AT DIFFERENT SALINITIES 118 ............. 4.2.2 EFFECT OF COMPATIBLE SOLUTES ON GROWTH OF S. XYLOSUS AND HALOMONAS HALO 121 ......................................................................... 4.2.3 BETAINE UPTAKE BY HALOMONAS HALO IN THE PRESENCE OR ABSENCE OF CCCP IN 0.5 M NaCl CHEMICALLY DEFINED MEDIUM. 124 4.2.4 BETAINE UPTAKE BY HALOMONAS HALO IN THE PRESENCE OR ABSENCE OF CCCP IN 3M NaCl CHEMICALLY DEFINED MEDIUM.... 127 4.2.5 BETAINE UPTAKE BY S. XYLOSUS IN THE PRESENCE OR ABSENCE OF CCCP IN 0.5 M NaCl CHEMICALLY DEFINED MEDIUM. 130 4.2.6 BETAINE UPTAKE BY S. XYLOSUS IN THE PRESENCE OR ABSENCE OF CCCP IN 3M NaCl CHEMICALLY DEFINED MEDIUM 130 .... V 4.2.7 PROLINE CONTENT